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Significance

Microbial arsenate respiration enhances the mobility of arsenic and contributes to the poisoning of tens of millions of people worldwide. Our ability to quantitatively predict how microbial activities shape arsenic geochemistry depends on a detailed understanding of how the enzymes that catalyze arsenate reduction work under environmentally relevant conditions. The structural and kinetic findings of the Arr enzyme complex reported here both help rationalize its extracytoplasmic localization and allow us to predict that the rate of arsenate release from minerals likely constrains its activity in sedimentary environments. Moreover, this work illustrates that engineering environmental bacteria to overexpress their native proteins can be straightforward, a strategy that may advance the study of enzymes that are challenging to express in traditional hosts.

Abstract

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit (Km = 44.6 ± 1.6 μM, kcat = 9,810 ± 220 seconds−1), and phosphate is a competitive inhibitor of arsenate reduction (Ki = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that (i) arsenate desorption limits microbiologically induced arsenate reductive mobilization and (ii) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.

Bacteria could help tackle the growing mountains of e-waste that plague the planet. Although researchers are a long way from optimizing the approach, some are already confident enough to pursue commercial ventures.

Holographic acoustic tweezers, in which ultrasonic waves produced by arrays of sound emitters are used to individually manipulate up to 25 millimeter-sized particles in three dimensions, could be used to create 3D displays consisting of levitating physical voxels.